Process and composition for treatment of luekemia and process for preparing the same

ABSTRACT

A process and composition are provided employing 10-ethyl- 10 deazaminopterin for treating leukemia, as well as other tumor systems including of ascitic character.

ORIGIN OF INVENTION

The invention described herein was in part made in the course of workunder a grant or award from the National Institute of Health, Departmentof Health, Education and Welfare.

This is a continuation of application Ser. No. 808,579, filed Dec. 13,1985, now abandoned, which in turn is a continuation of Ser. No.754,370, filed July 11, 1985, now abandoned, which in turn is acontinuation of Ser. No. 701,885, filed Feb. 15, 1985, now abandoned,which in turn is a continuation of Ser. No. 645,387, filed Aug. 29,1984, now abandoned, which in turn is a continuation of Ser. No.513,254, filed July 12, 1983, now abandoned, which in turn is acontinuation of Ser. No. 397,274, filed July 12, 1982, now abandoned,which in turn is a continuation of Ser. No. 75,913, filed Sept. 17,1979, now U.S. Pat. No. 4,393,064, patented July 12, 1983, which in turnis a continuation-in-part of Ser. No. 883,627, filed Mar. 6, 1978, ofSer. No. 761,152, filed Jan. 21, 1977, and of Ser. No. 664,213, filedMar. 5, 1976, all three now abandoned.

Leukemia is an acute or chronic disease of unknown cause in man andother warm-blooded animals. It is characterized by an abnormal increasein the number of immature leukocytes in the tissues of the body and inthe circulating blood. The disease apparently affects the blood-formingorgans, and is classified according to the type of leukocyte that isbeing proliferated abnormally. The disease is one of a number of formsof neoplastic disease, and the development of drugs for amelioration orcuring the disease has occupied the attention of research organizationsfor many years, and until most recently without appreciable success.Today, many forms of leukemia can be effectively treated with drugs. Inthe case of combination chemotherapy with acute lymphocytic leukemia inchildren a large percentage (50-60%) of five year survivals areobtained, and the disease is now classified as curable.

In accordance with the invention, it has been determined that leukemia,as well as other malignancies, including ascitic tumors, can beameliorated in warm-blooded lower animals and humans by theadministration of 10-ethyl-10-deazaminopterin.

The 10-ethyl-10-deazaminopterin of the invention has the structure:##STR1##

The relationship between N-ethyl-N-deazaminopterin and the N-10 methylderivative of aminopterin, methotrexate, is apparent from the following:##STR2##

4-Amino-4-deoxy-10-deazapteroic acid, a key intermediate for synthesisof 10-deazaminopterin, was first prepared by DeGraw, Brown, Kisliuk andGaumont, Journal of Medicinal Chemistry 14 866 (1971). DeGraw,Tsakotellis, Kisliuk, and Gaumont, Journal of Heterocyclic Chemistry 8105 (1971) had reported the potent growth -inhibitory activity of10-deazapteroic acid and its tetrahydro derivative against Streptococcusfaecium, a folate-dependent organism. Activity was greatly enhanced byreduction to the tetrahydro compound. Accordingly, it was thought thatthe 2,4-diamino-pteridines should be investigated, because they would beexpected to be more capable of cell penetration, and among the2,4-diamino-pteridines prepared was 4-amino-4-deoxy-10-deazapteroicacid, the compound shown under the Scheme I, Series d, at page 867 ofthe article.

In the Journal of Medicinal Chemistry 17 552 (1974) DeGraw, Kisliuk,Gaumont, Baugh and Nair reported on the synthesis and antifolateactivity of 10-deazaminopterin. The antimicrobial and antitumoractivities of the powerful dihydrofolic reductase inhibitors aminopterinand its N-10 methyl derivative, methotrexate, are well known, andnumerous analogues have been made to further improve the potency, cellpenetration and toxicity properties of these compounds. As part of acontinuing program, to investigate structure-activity relationships infolic acid analogues, DeGraw et al were interested in the effects ofreplacement of the nitrogen atom in the side chain of aminopterin, andreported on the synthesis and biological activity of 10-deazaminopterinin this paper. Continuing work with 10-deazaminopterin and its 10-alkylderivatives has now led to the discovery of their antileukemic activity,and to their efficacy in treating various ascites tumor systems, whichare the subject of this invention.

The process of treating leukemia and ascitic tumors according to thisinvention comprises administering to a warm-blooded animal having anabnormal proportion of leukocytes or other evidences of the malignancy,a therapeutic nontoxic amount of 10-ethyl-10-deazaminopterin as definedabove, as such or in the form of a pharmaceutically acceptable saltthereof. These salts are formed with one or more free NH₂ groups of the10-ethyl-10-deazaminopterin.

The process for preparing 10-ethyl-10-deazaminopterin is a synthesisincluding the following steps, starting from methoxymethyl acetylene(methyl propargyl ether): ##STR3##

Stage 1 is essentially a rearrangement of methyl propargyl ether to thecorresponding 1-methoxy-allene. The rearrangement proceeds underanhydrous conditions, in the presence of alkali, such as, for example,an alkali metal alkoxide, or inorganic alkali or alkali metal with theaddition of alcohol. Any lower alkanol such as methyl alcohol, ethylalcohol, propyl alcohol and butyl alcohol, and the iso, secondary andtertiary isomers thereof can be used as the alkoxide. Sodium andpotassium are the preferred alkali metals. The reaction proceeds atelevated temperature, and the use of reflux is a convenient method forcontrolling temperature. Oxygen should be excluded from the reactionmixture, in order to avoid reaction with the unsaturated groups. Aninert atmosphere such as nitrogen can be used.

In Stage 2, hydrogen chloride is added across the allenic double bondsof 1-methoxy-allene. Any alkoxy allene can be used, such as1-ethoxy-allene. The reaction proceeds under anhydrous conditions, atlow temperatures. The addition is made at low temperature, below about-25° C., and desirably below -70° C., to give ##STR4## but then thetemperature is increased to 0° C. to cause rearrangement to CH₃OCH═CHCH₂ Cl. This reaction also requires exclusion of oxygen, and itcan therefore be carried out under an inert atmosphere, such asnitrogen. A solution of anhydrous HCl in an inert solvent such asdiethyl ether is added slowly to the 1-methoxy-allene solution in thesame solvent. The reaction mixture can be used directly in Stage 3.

In Stage 3, the C10 carbon atom of the benzoic acid group of the10-deazaminopterin compound is alkylated by the3-chloro-1-methoxy-propene from Stage 2. The alkylation process requiresprereaction of a p-alkylbenzoic acid with a lithium alkylamide catalystin order to form an active anion reagent. The p-alkyl group has thestructure: ##STR5## corresponding to the C10 group of the 10-DA with afree hydrogen atom. This process (which also causes ionization of thecarboxyl group proton) requires lower temperatures at which it proceedsslowly, for example up to thirty hours. The reaction is preferablycarried out under anhydrous conditions under nitrogen in the presence ofan inert polar solvent such as tetrahydrofuran. The presence ofhexamethylphosphoric triamide (HMPA) cosolvent accelerates theionization process. After formation of the active anion, the3-chloro-1-methoxy-propene can then be added slowly to the anion reagentreaction mixture, again at room temperature or below. Since the reactiveanion is highly colored, and the reaction product is colorless, thereaction can be followed by discharge of color, and is complete when thereaction mixture is colorless. The solvents can then be removed, and thereaction product worked up.

In Stage 4 of the synthesis, a bromo-acetaldehyde group is formed bybromination and hydrolysis of the methoxy propylene substituent of theStage 3 reaction product. This reaction proceeds at a pH within therange from about 7 to about 9. Since the aqueous reaction solution fromStage 3 is alkaline, it is carbonated with gaseous carbon dioxide, toreduce the pH to within the range from about 8 to about 9. The reactionmay be conducted at a temperature within the range from about 0° toabout 50° C., but is preferably conducted at 0° to 5° C.

The reaction proceeds with bromine addition at the ethylene group, andhydrolytic conversion of the bromo methoxide to an aldehyde group, withloss of the HBr. The presence of an inert solvent, such asdichloro-methane, facilitates dispersal of the bromine and observationof discharge of bromine color. It is complete when the uptake of brominereaches the theoretical for the addition of one atom of bromine to theStage 3 reaction product. From time to time, addition of a buffer suchas solid sodium bicarbonate may be required to maintain the pH above 7.The reaction mixture is then acidified by addition of aqueoushydrochloric acid to a pH of about 2. The reaction mixture is worked upby extraction into an organic solvent and removal of solvent, giving thecrude bromoacetaldehyde acid reaction product whose formula is given inthe synthesis above.

In Stage 5, this reaction product is reacted with2,4,5,6-tetraaminopyrimidine in aqueous acetic acid, cyclizing the5,6-amino groups thereof with the aldehyde and bromine groups of theStage 4 reaction product, and forming a dihydro pteridine ring. Thepteridine ring is then dehydrogenated in Stage 6 forming4-amino-4-desoxy-10-deazaopteroic acid by reaction with potassiumiodide/iodine KI₃.

The 2,4,5,6-tetraaminopyrimidine sulfate used as the starting materialin Stage 5 is first reacted with barium chloride and water to remove thesulfate group, resulting in a barium sulfate precipitate. This can beremoved by filtration, and the filtrate can then be used in the Stage 5reaction.

The Stage 5 cyclization reaction proceeds at an acid pH, preferablywithin the range from 3 to 5, and consequently an acidic solvent such asaqueous acetic acid can be used. Aqueous acetic acid provides an acidicpH and the organic co-solvent effect of acetic acid aids in solubilizingthe bromo aldehydes. The reaction proceeds at moderately elevatedtemperatures within the range from 35° to 75° C., resulting in theformation of the dihydro pteridine.

This reaction product in the Stage 6 reaction is dehydrogenated withKI₃, obtained by dissolving iodine in an aqueous solution of potassiumiodide. The KI₃ can be added slowly to the dihydro pteridine, and thereaction can be followed by decolorization of the KI₃ solution. Whendecolorization ceases, the reaction is complete. Other suitableoxidizing agents such as hydrogen peroxide or potassium permanganate maybe used.

The reaction product is insoluble in the reaction mixture, and separatesout. In working up, the material can be filtered, washed and then dried.If desired, the precipitate can be taken up in dilute ammoniumhydroxide, and then re-precipitated with dilute acid, such as aceticacid.

The resulting 4-amino-4-desoxy-10-deazapteroic acid is then converted to10-ethyl-10-deazaminopterin in two steps, Stages 7 and 8. First, theproduct is reacted with isobutyl chloroformate, and then thediethyl-L-glutamate, converting the pteroic acid group to thecorresponding glutamide, diethyl ester, and the esterifying ethyl groupsare then hydrolyzed by reaction with dilute aqueous alkali, such asaqueous sodium hydroxide, forming the glutamide free diacid group of10-ethyl-10-deazaminopterin.

The Stage 7 reaction requires an acid acceptor to take up the liberatedhydrogen chloride. The Stage 7 reaction may be conducted with otheralkyl chloroformates such as methyl, ethyl, etc. Acid acceptors arepreferably organic bases such as tertiary amines or substitutedpyridines, for example, triethylamine, tributylamine,N-methylmorpholine, collidine and lutidine. The diethyl glutamate may beadded as the free base or as the hydrochloride salt in the presence ofan additional equivalent of the acid acceptor.

The reaction proceeds at room temperature or below, preferably 0° to -5°C., and an inert solvent can be used. The isobutyl chloroformate can beadded slowly to the reaction mixture, and upon completion of thereaction, diethyl-L-glutamate, organic amine and more solvent can beadded, and reaction continued at the same temperature until complete.

The reaction mixture is worked up by removing the solvent byevaporation, preferably in vacuo, and stirring the residue with a mildlyalkaline aqueous solution, such as aqueous sodium bicarbonate. Thediester is insoluble, and can be recovered by filtration, whileunreacted pteroic acid dissolves in the alkaline solution.

Hydrolysis of the esterifying ethyl groups is carried out with aqueousalkali at room temperature or above. The diester can be dissolved in asuitable solvent, such as 2-methoxyethanol, and held in the presence ofthe aqueous alkali until hydrolysis is complete. The acidic10-ethyl-10-deazaminopterin is soluble in aqueous alkali, and can thenbe precipitated by addition of acid, such as glacial acetic acid. Theprecipitate can be recovered, washed and dried.

The following Examples illustrate application of the synthesis to thepreparation of 10-ethyl-10-deazaminopter.

1-Methoxy-allene

The mixture of 100 ml of the methyl propargyl ether (1, dried over 4Asieves) and 3 g of potassium t-butoxide was stirred at reflux for fourhours under a very slight flow of nitrogen. The infrared spectrumindicated the rarrangement to be essentially complete. The liquid wasdistilled through a short path apparatus into a dry ice-cooled receiverto afford 72 g of 1-methoxy-allene; ir 1950 and 850 cm⁻¹.

3-Chloro-1-methoxypropene

A stream of dry HCl was passed into 700 ml of anhydrous ether chilled inan ice bath. After 45 g of HCl had been added another 267 ml of freshether was added. An aliquot of 541 ml (25.2 g, 0.69 mole HCl) was thenadded dropwise under nitrogen to a solution of 48.2 g (0.69 mole) of 1methoxy-allene in 240 ml of ether at -78° C. The addition required 2.5hours and the internal temperature was kept below -70° C. After thirtyminutes the solution was stored in a refrigerator at 0° to 5° C. fortwenty-four hours and was used directly in the next step.

4-Amino -4-desoxy-10-ethyl-10-deazapteroic acid

A solution of 192.8 ml (1.38 mole) of freshly distilled diisopropylamine in 1928 ml of dry THF was chilled to 0° to 5° C. and a solution of862 ml (1.38 mole) of 1.6M butyl lithium in hexane was added dropwisekeeping the temperature around 0° to 5° C. The mixture was stirredanother thirty minutes and a solution of 113 g (0.69 mole) of dryp-propyl benzoic acid (R₁ =H, R₂ =C₂ H₃) in 385 ml of dry THF was addeddropwise at 0° to 5° C. The red mixture was stirred at this temperaturefor 3.5 hours and then kept at 0° to 5° C. in a cold room fortwenty-five hours. The ether solution of 3-chloro-1-methoxypropene abovewas then added dropwise over 1.5 hour at 0° to 5° C.; quenching the redcolor was completed at the end of the addition period. After two hoursthe solvents were removed in vacuo and the residue partitioned between 1liter of water and 1 liter of ether. The aqueous portion was chilled,treated with gaseous CO₂ until it was pH 8 to 9, and 240 ml of CH₂ Cl₂was added. Then, at 0° to 5° C. was added, dropwise with stirring, 1MBr₂in CH₂ Cl₂ until persistence of the red color (85% uptake of Br₂observed). Solid NaHCO₃ was occasionally added to keep the pH at 7 to 8.The mixture was acidified to pH˜2 with 6N HCl (˜50 ml). The CH₂ Cl₂layer was removed and the aqueous extracted with another 200 ml of CH₂Cl₂. The combined organic extracts were dried over MgSO₄ and evaporatedin vacuo to leave an orange syrupy residue of the crude bromoaldehydeacid.

A mixture of 156.2 g (0.66 mole) of 2,4,5,6-tetraamino pyrimidinesulfate (Aldrich), 160.3 g (0.65 mole) of BaCl₂.2H₂ O and 3 liters ofwater was stirred at room temperature for 1.5 hours. The mixture waswarmed to 70° C., filtered hot and the filtrate cooled to roomtemperature. The filtrate was adjusted to pH 3 to 4 with 10% NaOH,warmed to 45° C. and treated over ten minutes with the bromoaldehydeabove in 1075 ml of glacial HOAc. The mixture was stirred at 50° for 1.5hours, decanted to remove some insoluble gum and cooled to roomtemperature. The solution containing the dihydro pteridine was treateddropwise with aqueous Kl₃ (from 81.9 g I₂ -156.2 g KI-1075 ml H₂ O)until decolorization of the KI₃. The solution was allowed to stand fortwenty-four hours and the yellow precipitate collected, washed withwater and ethanol and dried. The material was stirred with 1800 ml of H₂O containing 20 ml of concentrated NH₄ OH for two hours and filtered.The filtrate was acidified with HOAc to precipitate the product, whichwas collected, washed with water and dried, to afford a 32% yield, whichanalyzed as follows after crystallization from methanol:

    ______________________________________                                                                           0.1                                                                 0.1 N--HCl                                                                              N--NaOH                                    Formula  Analyzed        λ max.                                                                           λ max.                              (MW)     Calculated                                                                              Found     NM(E)   NM(E)                                    ______________________________________                                        C.sub.17 H.sub.18 N.sub.6 O.sub.2                                                      C 59.51   C 59.83   242 (22,622)                                                                          255 (26,281)                             2/5 CH.sub.3 OH                                                                        H 5.62    H 5.39                                                     (351.178)                                                                              N 23.93   N 23.62   338 (6,568)                                                                           370 (6,398)                              ______________________________________                                    

10-Ethyl-10-deazaminopterin

The mixture of 19.6 g (0.058 mole) of the pteroic acid, 15.8 ml oftriethylamine (0.116 mole) and 900 ml of dry DMF was warmed to 80° C.with stirring until nearly complete solution was obtained. The solutionwas cooled to 0° to 5° C. in an ice bath and treated dropwise with 15.0ml of isobutyl chloroformate (0.116 mole). After 1.5 hour at 0° to 5° C.the mixture was treated with a mixture of 28.0 g (0.116 mole) ofdiethyl-L-glutamate hydrochloride, 15.8 ml of triethylamine (0.116 mole)and 100 ml of dry DMF. The mixture was stirred for two hours in the icebath and at room temperature for twenty-four hours.

Dimethylformamide was evaporated under reduced pressure (˜1 mm) and theresidue was stirred with 400 ml 5% NaHCO₃ and 400 ml of ether for onehour. The mixture was filtered and the cake washed with water and etherand dried to leave the diester.

The diester was dissolved in 200 ml of 2-methoxy ethanol and treatedwith 100 ml of 1N NaOH. The solution was kept at room temperature for4.5 hours and then diluted with 1 liter of H₂ O. The solution wasacidified with glacial HOAc until precipitation was complete. Theprecipitate was collected by filtration. The cake was resuspended inwater with stirring, filtered and dried to leave crude10-ethyl-10-deazaminopterin; HPLC indicated 90% 10-ethyl-10-deazaminopterin and 10% of the unreacted pteroic acid. The yield was30%.

After purification by preparative HPLC the product analyzed as follows:

    ______________________________________                                                                           0.1                                                                 0.1 N--HCl                                                                              N--NaOH                                    Formula  Analyzed        λ max.                                                                           λ max.                              (MW)     Calculated                                                                              Found     NM(E)   NM(E)                                    ______________________________________                                        C.sub.22 H.sub.25 N.sub.7 O.sub.5                                                      C 52.95   C 53.43   242 (29,134)                                                                          255 (30,731)                             7/4 H.sub.2 O                                                                          H 5.41    H 5.43                                                     (494.515)                                                                              N 19.63   N 19.23   338 (10,102)                                                                          370 (7,582)                              ______________________________________                                    

The 10-ethyl-10-deazaminopterin can be administered per se, or inassociation with a pharmaceutically acceptable diluent or carrier. Theinvention accordingly also provides a pharmaceutical composition indosage unit form comprising from 0.1 to about 500 mg of10-ethyl-10-deazaminopterin, per dosage unit, together with apharmaceutically acceptable nontoxic inert carrier or diluent therefor.

The 10-ethyl-10-deazaminopterin can be used as such, or in the form ofan acid addition salt. These salts are formed with one or more free NH₂groups of the 10-deazaminopterin molecule.

The acid addition salts are preferably the pharmaceutically acceptable,nontoxic addition salts with suitable acids, such as those withinorganic acids, for example, hydrochloric, hydrobromic, nitric,sulphuric and phosphoric acids, and with organic acids, such as organiccarboxylic acids, for example, glycolic, maleic, hydroxymaleic, malic,tartaric, citric, salicylic, o-acetyloxybenzoic, nicotinic andisonicotinic acid, and organic sulphonic acids, for example,methanesulphonic, ethanesulphonic, 2-hydroxyethanesulphonic,toluene-p-sulphonic, and naphthalene-2-sulphonic acid. An acid additionsalt can be converted into the free compound according to known methods,for example, by treating it with a base, such as with a metal hydroxideor alkoxide, for example, an alkali metal or alkaline earth metalhydroxide, for example, lithium hydroxide, sodium hydroxide, potassiumhydroxide or calcium hydroxide; with a metal carbonate, such as analkali metal or an alkaline earth metal carbonate or hydrogen carbonate,for example, sodium, potassium or calcium carbonate or hydrogencarbonate; with ammonia; or with a hydroxyl ion exchange resin, or withany other suitable reagent.

An acid addition salt may also be converted into another acid additionsalt according to known methods; for example, a salt with an inorganicacid may be treated with a metal salt, for example a sodium, barium orsilver salt, of an acid in a suitable diluent, in which a resultinginorganic salt is insoluble and is thus removed from the reactionmedium. An acid addition salt may also be converted into another acidaddition salt by treatment with an anion exchange preparation.

The 10-ethyl-10-deazaminopterin or salt thereof can be administered tothe animal by any available route, including oral and parenteral(intravenous, intraperitoneal, subcutaneous, and intramuscularadministration. The amount administered is sufficient to ameliorate theleukemia or the ascitic tumor, and will depend upon the type ofleukemia, the species of animal, and the weight of the animal. Forexample, in human administration, a dosage of 10-deazaminopterincompound in within the range from about 0.1 mg/kg to about 500 mg/kg perday should be sufficient. Dosages in the higher part of the range,approaching 500 mg/kg, are normally administered in conjunction withleucovoran (dl-5-formyl tetrahydrofolate) to reduce toxicity. In thetreatment of lower test animals, a similar dosage range is therapeutic.The upper limit of dosage is that imposed by toxic side effects, and canbe determined by trial and error for the animal to be treated, includinghumans.

To facilitate administration, the 10-ethyl-10-deazaminopterin or saltthereof can be provided in composition form, and preferably in dosageunit form. While the compound can be administered per se, it is normallyadministered in conjunction with a pharmaceutically acceptable carriertherefor, which dilutes the compound and facilitates handling. The term"pharmaceutically acceptable" means that the carrier (as well as theresulting composition) is sterile and nontoxic.

The carrier or diluent can be solid, semisolid, or liquid, and can serveas a vehicle, excipient, or medium for the 10-ethyl-10-deazaminopterin.Exemplary diluents and carriers include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineraloil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin,syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl-and propyl-hydroxybenzoate, talc or magnesium stearate.

For convenience in handling, the 10-ethyl-10-deazaminopterin and carrieror diluent can be enclosed or encapsulated in a capsule, sachet, cachet,gelatin, paper or other container, especially when intended for use indosage units. The dosage units can for example take the form of tablets,capsules, suppositories or cachets.

The following Examples illustrate various forms of dosage units in whichthe 10-ethyl-10-deazaminopterin or salts thereof can be prepared:

EXAMPLE 1

    ______________________________________                                        Tablet formulation                                                                              Mg/tablet                                                   ______________________________________                                        10-Ethyl-10-Deazaminopterin                                                                       15                                                        Lactose             86                                                        Corn starch (dried) 45.5                                                      Gelatin             2.5                                                       Magnesium stearate  1.0                                                       ______________________________________                                    

The 10-ethyl-10-deazaminopterin is powdered and passed through a meshsieve and well mixed with the lactose and 30 mg of the corn starch, bothpassed through a sieve.

The mixed powders are massed with a warm gelatin solution, prepared bystirring the gelatin in water and heating to form a 10% w/w solution.The mass is granulated by passing through a sieve, and the moistgranules dried at 40° C.

The dried granules are regranulated by passing through a sieve and thebalance of the starch and the magnesium stearate is added and thoroughlymixed.

The granules are compressed to produce tablets each weighing 150 mg.

EXAMPLE 2

    ______________________________________                                        Tablet formulation                                                                              Mg/tablet                                                   ______________________________________                                        10-Ethyl-10-Deazaminopterin                                                                       100                                                       Lactose             39                                                        Corn starch (dried) 80                                                        Gelatin             4.0                                                       Magnesium stearate  2.0                                                       ______________________________________                                    

The method of preparation is identical with that of Example 1 exceptthat 60 mg of starch is used in the granulation process and 20 mg duringtabletting.

EXAMPLE 3

    ______________________________________                                        Capsule formulation                                                                             Mg/capsule                                                  ______________________________________                                        10-Ethyl-10-Deazaminopterin                                                                       250                                                       Lactose             150                                                       ______________________________________                                    

The 10-ethyl-10-deazaminopterin and lactose are passed through a sieveand the powders well mixed together before filling into hard gelatincapsules of suitable size, so that each capsule contains 400 mg of mixedpowders.

EXAMPLE 4

    ______________________________________                                        Suppositories                                                                                  Mg/suppositories                                             ______________________________________                                        10-Ethyl-10-Deazaminopterin                                                                      50                                                         Oil of Theobroma   950                                                        ______________________________________                                    

The 10-ethyl-10-deazaminopterin is powdered and passed through a sieveand triturated with molten oil of theobroma at 45° C. to form a smoothsuspension.

The mixture is well stirred and poured into molds, each of nominal 1 gcapacity, to produce suppositories.

EXAMPLE 5

    ______________________________________                                        Cachets                                                                                         Mg/cachet                                                   ______________________________________                                        10-Ethyl-10-Deazaminopterin                                                                       100                                                       Lactose             400                                                       ______________________________________                                    

The 10-ethyl-10-deazaminopterin is passed through a mesh sieve, mixedwith lactose previously sieved and fitted into cachets of suitable sizeso that each contains 500 mg.

EXAMPLE 6

    ______________________________________                                        Intramuscular injection                                                       (sterile suspension in aqueous vehicle)                                                                  Mg                                                 ______________________________________                                        10-Ethyl-10-Deazaminopterin  10                                               Sodium citrate               5.7                                              Sodium carboxymethylcellulose (low viscosity grade)                                                        2.0                                              Methyl para-hydroxybenzoate  1.5                                              Propyl para-hydroxybenzoate  0.2                                              Water for injection to 1.0 ml                                                 ______________________________________                                    

EXAMPLE 7

    ______________________________________                                        Intraperitoneal intraveneous or subcutaneous                                  injection (sterile solution in aqueous carrier                                system)                                                                                                  Mg                                                 ______________________________________                                        10-Ethyl-10-Deazaminopterin, hydrochloric                                                                  15                                               acid addition salt                                                            Sodium citrate               5.7                                              Sodium carboxymethylcellulose (low viscosity grade)                                                        2.0                                              Methyl para-hydroxybenzoate  1.5                                              Propyl para-hydroxybenzoate  0.2                                              Water for injection to 1.0 ml                                                 ______________________________________                                    

The following Example illustrates administration of10-ethyl-10-deazaminopterin using a standardized test procedure:

EXAMPLE 8

Sodium hydroxide (0.2 ml of 0.1N) was added to 5 mg of10-ethyl-10-deazaminopterin. Distilled water was then added, the pHadjusted to 7.0, and the solution then diluted with distilled water to10 ml.

The resulting solution and dilutions thereof were administered inaliquots of 0.1 ml by intraperitoneal injection into L1210 leukemic BD(2) F₁ female mice (A. R. Schmid, Madison, Wis.). Injections were givenonce per day, three times per week (Monday, Wednesday, Friday) startingone day after tumor transplantation (10⁶ cells/mouse). Therapy wascontinued until death of the animals.

For comparison purposes, and as a control, a parallel series of testswas carried out simultaneously using L1210 leukemic BD (2) F₁ femalemice, under exactly the same test conditions, administering methotrexateinstead of 10-ethyl-10-deazaminopterin.

The procedure for testing, and the maintenance and transplantation ofthe L1210 leukemia, is in accordance with the method of Hutchinson, D.J., Robinson, D. C., Martin, D., Ittensohn, O. L. and Dillenberg,Journal Cancer Res. 22 57-72 (1962). The antileukemic activity of10-ethyl-10-deazaminopterin was evaluated against methotrexate in termsof the increase in median life span obtained at various dosages, up tothe maximum tolerated level, when compared to untreated controls.Toxicity of various dosages was evaluated by the extent of weight lossand eventual death, with no evidence of tumor.

Representative results obtained against the L1210 leukemia are asfollows:

                  TABLE                                                           ______________________________________                                        L1210 Leukemia:                                                               Inoculum:     10.sup.6 cells                                                  Host:         BD (2) F.sub.1 female mice                                      Vehicle:      Buffered isosaline                                              Schedule:     Every two days times five                                                                      8                                                                             10-ethyl-                                      Mice            Methotrexate   10-deazaminopterin                             (Exp's ×                                                                       Dosage   AST       ILS    AST     ILS                                  No.)   (mg/kg)  days      %      days    %                                    ______________________________________                                        5 × 2                                                                          --        6.7 ± 0.7                                                                           --      6.7 ± 0.7                                                                         --                                   5 × 2                                                                           6       13.8 ± 1.2                                                                           +105                                                5 × 2                                                                           9       15.8 ± 1.7                                                                           +135                                                5 × 2                                                                          12       16.9 ± 1.5                                                                           +149   18.3 ± 1.7                                                                         +172                                 5 × 2                                                                          18       17.3 ± 1.8                                                                           +158   21.8 ± 1.4                                                                         +220                                 5 × 2                                                                          24       15.3 ± 2.1                                                                           +127                                                ______________________________________                                    

It is apparent from the above results that the life span of the testmice was very considerably increased by administration of10-ethyl-10-deazaminopterin. Increased dosages led to increased lifespans, the maximum life span being obtained with the maximum dose, about22 mg/kg, at which dosage level a slight toxicity was observed, asevidenced by a decrease in weight change. The results show10-ethyl-10-deazaminopterin on a similar dosage level to be superior tomethotrexate, and since methotrexate is accepted as effective,10-ethyl-10 deazaminopterin is to be expected to be at least aseffective as methotrexate under similar conditions, and probablyslightly more powerful, dosagewise. The potent antileukemic activity of10-ethyl-10-deazaminopterin is evident from these test results.

Having regard to the foregoing disclosure, the following is claimed asinventive and patentable embodiments thereof:
 1. A pharamceuticalcomposition in tablet form for treating leukemia or an ascites tumorcomprising an amount within the range from about 0.1 to about 500 mg of10-ethyl-10-deazaminopterin per tablet therapeutically effective toameliorate leukemia or the ascites tumor, together with apharmaceutically acceptable nontoxic carrier or diluent therefor.
 2. Apharmaceutical composition according to claim 1 in which the10-ethyl-10-deazaminopterin is in the form of a pharmaceuticallyacceptable acid addition salt or quaternary ammonium derivative.
 3. Apharmaceutical composition in capsule form for treating leukemia or anascites tumor comprising an amount within the range from 0.1 to about500 mg of 10-ethyl-10-deazaminopterin per capsule therapeuticallyeffective to ameliorate leukemia or the ascites tumor, together with apharmaceutically acceptable nontoxic inert carrier or diluent therefor.4. A pharmaceutical composition according to claim 3 in which the10-ethyl-10-deazaminopterin is in the form of a pharmaceuticallyacceptable acid addition salt or quaternary ammonium derivative.
 5. Apharmaceutical composition in suppository form for treating leukemia oran ascites tumor comprising an amount within the range from about 0.1 toabout 500 mg of 10-ethyl-10-deazaminopterin per suppositorytherapeutically effective to ameliorate leukemia or the ascites tumor,together with a pharmaceutically acceptable nontoxic inert carrier ordiluent therefor.
 6. A pharmaceutical composition according to claim 5in which the 10-ethyl-10-deazaminopterin is in the form of apharmaceutically acceptable acid addition salt or quaternary ammoniumderivative.
 7. A pharmaceutical composition in cachet form for treatingleukemia or an ascites tumor comprising an amount within the range fromabout 0.1 to about 500 mg of a 10-ethyl-10-deazaminopterin per cachettherapeutically effective to ameliorate leukemia or the ascites tumor,together with a pharmaceutically acceptable nontoxic inert carrier ordiluent therefor.
 8. A pharmaceutical composition according to claim 7in which the 10-ethyl-10-deazaminopterin is in the form of apharmaceutically acceptable acid addition salt or quaternary ammoniumderivative.
 9. A pharmaceutical composition in sterile aqueous form fortreating leukemia or an ascites tumor comprising an amount within therange from about 0.1 to about 500 mg of 10-ethyl-10-deazaminopterintherapeutically effective to ameliorate leukemia or the ascites tumor,together with a pharmaceutically acceptable nontoxic sterile inertaqueous carrier or diluent therefor.
 10. A pharmaceutical compositionaccording to claim 9 in which the 10-ethyl-10-deazaminopterin is in theform of a pharmaceutically acceptable acid addition salt or quaternaryammonium derivative.
 11. A pharmaceutical composition according to claim9 in aqueous solution form.
 12. A pharmaceutical composition accordingto claim 9 in aqueous dispersion form.
 13. A process for treatingleukemia and ascites tumors which comprises administering to awarm-blooded animal having an abnormal proportion of leukocytes or otherevidence of the malignancy, a therapeutic and relatively nontoxic amountof 10-ethyl-10-deazaminopterin.
 14. A process according to claim 13 inwhich the 10-ethyl-10-deazaminopterin is administered as apharmaceutically acceptable salt thereof.
 15. A process according toclaim 13 in which the 10-ethyl-10-deazaminopterin is administered in anamount within the range from about 0.1 to about 500 mg per day.
 16. Aprocess according to claim 13 in which the 10-ethyl-10-deazaminopterinis administered with an inert diluent or carrier.
 17. A processaccording to claim 13 in which the 10-ethyl-10-deazaminopterin isadministered orally.
 18. A process according to claim 13 in which the10-ethyl-10-deazaminopterin is administered parenterally.